Development of species‐specific PCR primers on rDNA for the identification of European Armillaria species

Attempts to design species‐specific PCR primers from six European Armillaria species in the ribosomal RNA genes are reported. Primers were developed on the basis of the nucleotide sequence variability of the internal transcribed spacers (ITS) and the intergenic spacer (IGS1) of the ribosomal DNA. Four sets of primers gave specific PCR products for Armillaria tabescens, Armillaria mellea and Armillaria ostoyae. However, due to the high sequence similarities between Armillaria borealis and Armillaria ostoyae and between Armillaria cepistipes and Armillaria gallica no species specific amplification was obtained for these taxa.     

Identification of Armillaria species in Japan using PCR-RFLP analysis of rDNA intergenic spacer region and comparisons of Armillaria species in the world

Armillaria species from Japan were characterized using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the intergenic spacer region-1 (IGS-1) of ribosomal DNA (rDNA). Eleven different digestion patterns by restriction endonuclease Alu I were found among 70 isolates of seven Armillaria species in Japan. Isolates within Armillaria nabsnona, A. ostoyae, A. cepistipes, and Japanese biological species E showed the same Alu I digestion patterns. Five Alu I patterns were detected for A. gallica, three patterns for A. mellea, and two patterns for A. tabescens. Seven Armillaria species in Japan were clearly distinguished by using the profiles obtained when PCR products were digested with Alu I, Msp I, and Hae III restriction enzymes. There was considerable variability of Alu I restriction sites within the IGS-1 between the isolates of five Armillaria species, A. gallica, A. nabsnona, A. cepistipes, A. mellea, and A. tabescens, in Japan and those of their European and North American counterparts.     

Ecology of Armillaria species in managed forests and plantations in Serbia

The distribution of Armillaria species was investigated in Serbian forest ecosystems, in relation to the main host species attacked, forest‐types, geography and altitude. In total, 388 isolates were identified from 36 host species in 47 sites. Armillaria gallica was the most commonly observed species with the widest distribution and with an altitudinal range of 70–1450 m, it was the dominating Armillaria species in lowland alluvial forests and in Quercus and Fagus forests at higher elevations. Armillaria mellea occurred in Quercus spp. – dominated forests in the north and central regions at 70–1050 m. Sixty‐eight per cent of the A. mellea isolates were collected from living hosts, most commonly in declining conifer plantations. Armillaria ostoyae was distributed in the cooler coniferous forest types and plantations in the Dinaric Alps in the south of Serbia, at 850–1820 m. Armillaria cepistipes was found in the eastern and southern hilly and mountainous regions of the country, at 600–1900 m. Most isolates were obtained from conifers and rhizomorphs in the soil around decaying stumps. Armillaria tabescens was found only on dead oak material in the northern and eastern regions of the country at altitudes lower than 600 m.     

Development and verification of a diagnostic assay based on EF‐1 α for the identification of Armillaria species in Northern Europe

The overall aim of this study was to develop a new, reliable and rapid diagnostic assay for differentiating six European Armillaria species based on variation in their elongation factor‐1 alpha (EF‐1 α) gene sequences and to verify a set of species‐specific primers on 61 Armillaria isolates from Europe. Partial sequences of the EF‐1 α gene obtained in Armillaria borealis, Armillaria cepistipes, Armillaria gallica, Armillaria mellea, Armillaria ostoyae and Armillaria tabescens revealed sufficient interspecific variation to distinguish among species using nested primers. These primers gave unambiguous bands when tested on representative isolates of five of these species. However, the EF‐1 α sequences of European A. borealis isolates clustered into two distinct clades, termed here AbX and AbY. Specific primers were subsequently designed and tested successfully on both AbX‐type and AbY‐type A. borealis isolates. The taxonomy of A. borealis needs to be elucidated to determine whether a new, as yet unnamed Armillaria taxon exists in Europe. Three A. borealis isolates were also found to have heterozygous sites in their EF‐1 α sequences, which suggests that the gene could exist in more than one copy or that these isolates contain hybrid sequences. A pyrosequencing method was also developed, targeting a small region of EF‐1 α intron 4, which was able to differentiate European Armillaria isolates to the species level and additionally could distinguish AbX‐type and AbY‐type A. borealis isolates.     

Ecology of Armillaria species on conifers in Japan

Distribution, host preference and pathogenicity of Japanese Armillaria species on conifers were investigated on the basis of field collections of 65 isolates. We identified seven Armillaria species from 19 conifer species including six major Japanese plantation conifers using mating tests and sequences of the translation elongation‐1 α gene. Armillaria mellea, Armillaria ostoyae, Armillaria cepistipes and Armillaria sinapina were frequently collected, whereas Armillaria nabsnona, Armillaria tabescens and a biological species Nagasawa’s E were rare. On the basis of host condition when the isolates were collected, A. mellea, A. ostoyae, A. cepistipes and A. tabescens are considered as moderate to aggressive pathogens of conifers in Japan.     

Phylogenetic analysis of Japanese Armillaria based on the intergenic spacer (IGS) sequences of their ribosomal DNA

To determine the phylogenetic positions of two new species, Armillaria jezoensis and Armillaria singula, and one new subspecies, Armillaria mellea suhsp. nipponica, the nucleotide sequences of the intergenic spacers (IGS) of their ribosomal DNA were investigated, and compared with those of tour other Armillaria species from Japan, and those of nine Armillaria species from Europe and North America. We conclude that Armillaria jezoensis, and Armillaria singula belong to the Armillaria gallica cluster as Armillaria cepistipes, Armillaria gallica and Armillaria sinapina from Japan. Two isolates of Armillaria ostoyae from Japan were placed within the Armillaria ostoyae cluster. Armillaria mellea subsp. nipponica had an IGS sequence as long as the IGS of Armillaria mellea from Europe and North America. However, the IGS sequences of Armillaria mellea subsp. nipponica, whose basidium base lacks a clamp connection could not be satisfactorily aligned with the IGS sequences of other species possessing this morphological feature.     

Characterization of North American Armillaria species: genetic relationships determined by ribosomal DNA sequences and AFLP markers

Phylogenetic and genetic relationships among 10 North American Armillaria species were analysed using sequence data from ribosomal DNA (rDNA), including intergenic spacer (IGS‐1), internal transcribed spacers with associated 5.8S (ITS + 5.8S), and nuclear large subunit rDNA (nLSU), and amplified fragment length polymorphism (AFLP) markers. Based on rDNA sequence data, the nLSU region is less variable among Armillaria species than the ITS + 5.8S and IGS‐1 regions (nLSU < ITS + 5.8S < IGS‐1). Phylogenetic analyses of the rDNA sequences suggested Armillaria mellea, A. tabescens and A. nabsnona are well separated from the remaining Armillaria species (A. ostoyae, A. gemina, A. calvescens, A. sinapina, A. gallica, NABS X and A. cepistipes). Several Armillaria species (A. calvescens, A. sinapina, A. gallica, NABS X and A. cepistipes) clustered together based on rDNA sequencing data. Based on the isolates used in this study, it appears that techniques based on IGS‐1, ITS + 5.8S, and/or D‐domain/3′ ends of nLSU are not reliable for distinguishing A. calvescens, A. sinapina, A. gallica and A. cepistipes. However, AFLP data provided delineation among these species, and AFLP analysis supported taxonomic classification established by conventional methods (morphology and interfertility tests). Our results indicate that AFLP genetic markers offer potential for distinguishing currently recognized North American Biological Species (NABS) of Armillaria in future biological, ecological and taxonomic studies.     

Distribution,ecology and host range of Armillaria species in Albania

Species of Armillaria were identified from 645 isolates obtained in a nation‐wide survey in Albania. The material was collected from ca. 250 permanent plots, established for monitoring forest health, and from forests and orchards attacked by Armillaria. Armillaria mellea s.s. occurred on several coniferous and broadleaved trees in most areas examined, although it was absent above 1100–1200 m in northern Albania. This species damaged Abies and Quercus spp. and, to a lesser extent, other forest trees. Armillaria mellea was also commonly recorded causing damage in orchards and vineyards. Armillaria gallica was a common saprophyte or weak pathogen in coniferous and deciduous forests at altitudes from 600 to 1600 m, and less commonly on oaks at lower altitudes. Armillaria ostoyae was rare in central and southern Albania, but common in northern Albania, causing significant damage to pine and other conifers, mostly at altitudes from 600 to 1800 m. Armillaria cepistipes was recorded at altitudes from 800 to 1800 m as a saprophyte or weak pathogen on conifers and deciduous trees, mostly in beech and silver fir forests. Armillaria tabescens was found in oak forests at altitudes from sea level to 900 m. In orchards, A. tabescens occasionally attacked almond and pear trees. Armillaria borealis was found in a few locations in northern Albania, at altitudes from 800 to 1800 m.     

Ecology and distribution of Armillaria species in Norway

The occurence of Armillaria species was assessed in Norway, enabling the northern‐most distribution of this genus to be determined in Europe. Four Armillaria species were found in Norway. Armillaria borealis was the most common species occurring on woody vegetation to the permafrost zone (ca. 69°N). Armillaria cepistipes was present in southern and central Norway, but was not found further than 66°N. Armillaria solidipes and Armillaria gallica were rare, found at only one locality each; 59°40′ and 59°32′, respectively. Armillaria species were found on 14 hosts, but there was no significant difference between occurrence of A. borealis and A. cepistipes on declining and dead trees. Phylogenetic analyses separated each species into separate clades. All isolates of A. borealis, except one, and most isolates of A. solidipes were in separate clades. However, a subclade within the A. borealis clade was formed of two A. ostoyae and one A. borealis isolates. Two small A. cepistipes genets were found in a declining oak stand.     

Laccase isoenzyme patterns of European Armillaria species from culture filtrates and infected woody plant tissues

Polyacrylamide isoelectric focusing with specific staining for laccase activity was used to characterize laccase from European Armillaria species (Armillaria ostoyae, Armillaria mellea, Armillaria gallica, Armillaria cepistipes). The enzyme was extracted from culture media either supplemented, or not, with pine sawdust, and also from Pinus pinaster naturally infected by A. ostoyae, or artificially inoculated with A. mellea and A. ostoyae. Some differences in banding patterns were found for Armillana isolates according to the species and the culture media, but a common band at pI = 3.4 was found in all the extracts tested, independently of their origin (culture filtrate or wood).     

Distribution and ecology of Armillaria species in Greece

Five Armillaria species were identified in a nationwide survey in Greece. Armillaria mellea was present in coniferous and broad-leaved forests in most of the areas examined, except the high altitudes (above 1100 m) of the mountains of north Greece. It was found to cause significant damage in fir forests as well as in fruit orchards and vineyards. Armillaria gallica was common in coniferous and broad-leaved forests in the high altitudes of central and northern Greece, predominating in the beech forests. The fungus was a weak parasite or a saprophyte of forest trees and was occasionally found on cultivated plants. Armillaria ostoyae was not found in southern and central parts of the country, but it has a wide distribution in the mountain forests of northern Greece and causes significant damage on fir, black pine, Scots pine and spruce. Armillaria cepistipes was recorded at high altitudes (1400–1800 m) on two mountains of northern Greece, mostly as a saprophyte in coniferous and broad-leaved forests. Armillaria tabescens was rare in the forests of Greece; it was found to cause disease in almond tree orchards.     

Armillaria in an ancient broadleaved woodland

In an ancient broadleaved woodland in eastern England that had been coppiced regularly for over 700 years Armillaria mellea and A. gallica were common, A. tabescens was moderately frequent, whilst A ostoyae and A cepistipes were local. Fruit bodies were often abundant in areas coppiced the previous winter. The largest focus of A gallica occupied 9 ha and was probably over 500 years old. Large trees were rarely killed but A mellea caused some root decay in birches. The number of coppice shoots killed by Armillaria was small but became greater as the period since coppicing increased; A gallica was most often involved. Alder coppice was the most, and hazel coppice the least, affected. The productivity of coppiced woodland in general seemed little reduced by Armilaria.     

Initial characterization of an unidentified Armillaria isolate from Serbia using LSU‐IGS1 and TEF‐1‐α genes

Armillaria species have a global distribution and play variable ecological roles, including causing root disease of diverse forest, ornamental and horticultural trees. Accurate identification of Armillaria species is critical to understand their distribution and ecological roles. This work focused on characterizing an unidentified Armillaria isolate from a Serbian forest using pairing, sequencing of the partial large subunit and intergenic spacer‐1 regions of rDNA (LSU‐IGS1) and the translation elongation factor‐1 alpha gene (tef‐1α) genes, and phylogenetic analyses. Despite previously obtained LSU‐IGS1 RFLP patterns that matched the newly described North American Armillaria altimontana, pairing tests and phylogenetic analyses of LSU‐IGS1 and tef‐1α sequences clearly demonstrate that the unidentified isolate is not A. altimontana. Based on LSU‐IGS1, Armillaria gallica isolates were polyphyletic, and the Serbian isolate clustered with a subset of European A. gallica isolates within a well‐supported clade (99%). Based on tef‐1α, the Serbian isolate appeared as a separate, well‐supported clade (97%) that was basal to other poorly resolved, polyphyletic clades containing European A. gallica isolates. It is speculated that the unidentified Armillaria isolate from Serbia could represent an evolutionary ancestral state because of its separate, basal position compared with other clades comprising polyphyletic European A. gallica isolates. Alternatively, this unidentified Serbian isolate could represent an unusual hybrid because of its high‐level sequence heterogeneity, represented by multiple two‐nucleotide codes, within tef‐1α. Further characterization is needed to confirm the taxonomic status and ecological/evolutionary significance of this unique, unknown Armillaria isolate from Serbia.     

Geographical distribution of the Armillaria species in The Netherlands in relation to soil type and hosts

The geographical distribution of the annulate Armillaria species was studied in The Netherlands during the period 1983–1992. Armillaria gallica (incl. A. cepistipes), A. mellea and A. ostoyae appear to be widespread, the first two species being rather common on broad-leaved hosts growing on clay and loess soils, and the third species common on both broad-leaved and coniferous hosts on acid sandy soils. The distribution of the Armillaria species encountered was primarily determined by soil type. From a silvicultural point of view, A. ostoyae is the most important species, being pathogenic and occurring most frequently on sandy soils, the soil type predominantly used for forestry in The Netherlands.     

Distribution of Armillaria species in upland Ozark Mountain forests with respect to site,overstory species composition and oak decline

Armillaria root disease is a contributing factor to oak decline in the Ozark Mountains of central USA. We have identified Armillaria gallica, Armillaria mellea, and Armillaria tabescens in Quercus‐Carya‐Pinus forests of the region. Presence/absence patterns of each Armillaria species as well as all possible Armillaria species combinations were analysed by contingency tables and/or stepwise logistic multiple regressions with principal characteristics of the studied sites and forest stands, both quantitative and qualitative: geographic land‐type association, bedrock type, landform position, slope direction (aspect), soil type and soil surface stone cover, down woody debris, abundance and basal area of woody vegetation and decline mortality by species. Most decline mortality consisted of two red oak species (section Erythrobalanus, Quercus coccinea and Quercus velutina), which also were most sensitive to Armillaria infection. Site characteristics related to the distributions of Armillaria species and decline mortality were also related to the preponderance of Q. coccinea and Q. velutina, regional vegetation history (i.e. conversion of Pinus echinata stands to hardwoods), and the different strategies of territory acquisition and spread of the Armillaria species involved. The presence of A. gallica may reduce the activity of more virulent Armillaria species.     

Comparison of the virulence of Armillaria cepistipes and Armillaria ostoyae on four Norway spruce provenances

The basidiomycetes Armillaria cepistipes and Armillaria ostoyae frequently occur in the same forest stand. In this study, we determined the virulence of 20 isolates of A. cepistipes and 16 isolates of A. ostoyae on four different provenances of 2‐year‐old Norway spruce (Picea abies). Within 30 months after inoculation, 1.1 and 19.1% of the seedlings inoculated with A. cepistipes and A. ostoyae, respectively, had died or were dying. The incidence of dead and dying seedlings varied between 3 and 49% among the A. ostoyae isolates. The virulence of an isolate was positively correlated to its ability to produce rhizomorphs. One Norway spruce provenance showed significantly lower susceptibility to A. ostoyae than the other three. Rhizomorphs of both Armillaria species were attached to the root surface. The attached rhizomorphs of A. ostoyae, however, were associated with significantly more lesions. The virulence of the isolates was not correlated with their wood‐degrading capability for either of the Armillaria species.     

Rhizomorph production and stump colonization by co‐occurring Armillaria cepistipes and Armillaria ostoyae: an experimental study

In managed spruce forests, Armillaria cepistipes and A. ostoyae are efficient stump colonizers and may compete for these resources when they co‐occur at the same site. The aim of this experiment was to quantify the mutual competitive ability of the two Armillaria species in producing rhizomorphs and in colonizing Norway spruce (Picea abies) stumps. Five isolates of A. cepistipes and two isolates of A. ostoyae were simultaneously inoculated pair‐wise into pots containing a 4‐year‐old spruce seedling. For comparison, each isolate was also inoculated alone. One year after inoculation, stumps were created by cutting down the seedlings. Six months after creation of the stumps, rhizomorph production and stump colonization were assessed. Armillaria spp. were identified from 347 rhizomorphs and 48 colonized stumps. Armillaria cepistipes dominated both as rhizomorphs in the soil and on the stumps. Nevertheless, A. ostoyae was relatively more frequent on the stumps than in the soil and A. cepistipes was relatively more frequent in the soil than on the stumps. In both species, the ability to colonize the stumps in simultaneous inoculations was significantly reduced compared with single inoculations. In respect to rhizomorph production, simultaneous co‐inoculations had a slightly stimulatory effect on A. cepistipes and no significant effect on A. ostoyae. Our study suggests a rather neutralistic co‐existence of A. cepistipes and A. ostoyae as rhizomorphs in the soil. Concerning the ability to colonize stumps, the two species experience a mutual negative effect from the interaction, probably because of interspecific competition.     

Armillaria species distribution on symptomatic hosts in northern hardwood and mixed oak forests in western Massachusetts

Armillaria spp. are some of the most important forest pathogens in mixed hardwood forests of southern New England, yet their role as prominent disturbance agents is still not fully appreciated. We investigated the distribution of Armillaria species across eight separate stands of northern hardwood and mixed oak forests in western Massachusetts. We were specifically interested in the Armillaria species distribution from live, symptomatic hosts and not in determining overall incidence in the forest. From 32 plots (16 within each forest type), 320 isolates were collected. Armillaria was routinely encountered causing disease of live trees. In total, 89% (286/320) of all isolations came from live hosts exhibiting symptoms of root and butt rot. Overall, A. gallica was the dominant species in each forest type, making up 88/160 (55%) isolates from northern hardwood and 153/160 (96%) of all isolations from mixed oak stands. However, northern hardwood forests showed much greater species diversity, as A. calvescens, A. gemina, A. ostoyae, and A. sinapina were all found. At one site, a northern hardwood forest surrounding a high elevation spruce-fir forest, A. ostoyae was the most abundant species encountered. All five Armillaria species were found causing disease of live hosts, including A. gemina, a species considered by some as weakly virulent. Armillaria gallica was found on 22/23 tree species’ sampled, and was found most often causing butt rot.     

Identification and distribution of Armillaria species associated with an oak decline event in the Arkansas Ozarks

Forests in the Ozark Mountains of northern Arkansas recently experienced a widespread oak decline event. Armillaria, a root rot fungus, has been associated with other oak decline events and may have been an important contributing factor to tree mortality in this event. Although Armillaria has been identified from the Ozark Mountains in Missouri, it has never been investigated in the Arkansas Ozarks. Molecular diagnostic techniques were used in this study to identify species of Armillaria present on roots removed from dead trees of two common oak species, northern red oak, Quercus rubra L., and white oak, Q. alba L., from three geographic areas and on three topographic positions – ridges, south‐ and west‐facing benches. Armillaria(A. mellea, A. gallica or A. tabescens) was identified from 31% of root samples taken from 102 trees in seven of nine sample plots. Armillaria mellea, occurred most often (20 samples, both oak species on seven plots) followed by A. gallica (10 samples, northern red oak only on four plots), and A. tabescens occurred twice (on northern red oak in a single plot). Thus, all three Armillaria species occurred on northern red oaks while A. mellea was the only species recovered from white oaks. Results varied by topographic position with samples from tree roots on ridges having the fewest positive identifications, one of 29. West‐facing benches had the highest positive samples with 20 of 41 testing positive and trees on south‐facing benches were intermediate with 11 of 32 samples from infected trees. This study documents the occurrence of three species of Armillaria in the Arkansas Ozarks and their association with oak mortality resulting from an oak decline event coupled with a red oak borer, Enaphalodes rufulus, outbreak. Further, it documents some potential variation in host/pathogen combinations and forest site conditions.     

Ecology of Armillaria species on silver fir (Abies alba) in the Spanish Pyrenees

? We describe the distribution and the ecology of three Armillaria species observed in silver fir (Abies alba) forests of the Pyrenees.

? We surveyed the presence and abundance of Armillaria above and belowground in 29 stands. Isolates were identified by the PCR-RFLP pattern of the IGS-1 region of their ribosomal DNA. We measured several ecological and management parameters of each stand in order to describe Armillaria infected sites.

? Armillaria cepistipes was the most abundant of three species observed. Armillaria gallica was dominant in soils with a higher pH and at lower elevations. Armillaria ostoyae seemed to be more frequent in stands where A. alba recently increased its dominance relative to other forest tree species. Thinning activities correlated with an increased abundance of Armillaria belowground. In 83% of the stands the same Armillaria species was observed above and belowground.

? It seems that in a conifer forest, A. cepistipes can be more frequent than A. ostoyae, a virulent conifer pathogen. Since logging is related to a higher abundance of Armillaria in the soil, the particular Armillaria species present in a given stand could be considered an additional site factor when making management decisions.